Multi-modal mechanisms of the metastasis suppressor, NDRG1: Inhibition of WNT/β-catenin signaling by stabilization of protein kinase Cα

The metastasis suppressor, N-myc downstream regulated gene-1 (NDRG1), inhibits pro-oncogenic signaling in pancreatic cancer (PC). This investigation dissected a novel mechanism induced by NDRG1 on WNT/β-catenin signaling in multiple PC cell types. NDRG1 overexpression decreased β-catenin and downregulated glycogen synthase kinase-3β (GSK-3β) protein levels and its activation. However, β-catenin phosphorylation at Ser33, Ser37, and Thr41 are classically induced by GSK-3β was significantly increased after NDRG1 overexpression, suggesting a GSK-3β-independent mechanism. Intriguingly, NDRG1 overexpression upregulated protein kinase Cα (PKCα), with PKCα silencing preventing β-catenin phosphorylation at Ser33, Ser37, and Thr41, and decreasing β-catenin expression. Further, NDRG1 and PKCα were demonstrated to associate, with PKCα stabilization occurring after NDRG1 overexpression. PKCα half-life increased from 1.5 ± 0.8 h (3) in control cells to 11.0 ± 2.5 h (3) after NDRG1 overexpression. Thus, NDRG1 overexpression leads to the association of NDRG1 with PKCα and PKCα stabilization, resulting in β-catenin phosphorylation at Ser33, Ser37, and Thr41. The association between PKCα, NDRG1, and β-catenin was identified, with the formation of a potential metabolon that promotes the latter β-catenin phosphorylation. This anti-oncogenic activity of NDRG1 was multi-modal, with the above mechanism accompanied by the downregulation of the nucleo-cytoplasmic shuttling protein, p21-activated kinase 4 (PAK4), which is involved in β-catenin nuclear translocation, inhibition of AKT phosphorylation (Ser473), and decreased β-catenin phosphorylation at Ser552 that suppresses its transcriptional activity. These mechanisms of NDRG1 activity are important to dissect to understand the marked anti-cancer efficacy of NDRG1-inducing thiosemicarbazones that upregulate PKCα and inhibit WNT signaling.

The anti-oncogenic and anti-metastatic activities of NDRG1 are mediated by its ability to inhibit a broad range of oncogenic pathways responsible for angiogenesis, tumor growth, and metastasis (5)(6)(7)(8)(9)(10).NDRG1 is composed of 394 amino acids, which includes multiple structural motifs including helix-turnhelix near the N-terminus, an inactive a/b hydrolase motif that encompasses a phosphopantetheine attachment site, and a cap-like domain, and three tandem repeats near the C-terminus (11,12).
WNT/b-catenin signaling is tightly regulated by the destruction complex consisting primarily of axin1, casein kinase 1a (CK1a), adenomatous polyposis coli (APC), and glycogen synthase kinase-3b (GSK-3b) (7,13).Classically, GSK-3b is involved in b-catenin phosphorylation at Ser33, Ser37, and Thr41 causing its destabilization and degradation by the proteasome (7,13).The destruction complex is regulated by the binding of WNT ligands to the frizzled and LRP5/ 6 receptors, with the disheveled protein binding to these receptors (7,13).The interaction of these receptors with disheveled leads to the dissociation of the destruction complex, resulting in the accumulation of cytoplasmic b-catenin and its nuclear translocation (7,13).Once b-catenin translocates to the nucleus, it acts as a transcriptional co-activator of transcription factors of the T cell factor and lymphoid enhancer factor-1 family that transactivate oncogenic target genes, such as those encoding c-Myc and cyclin D1 (20,21).
Aberrant WNT/b-catenin signaling has been demonstrated in many cancers such as pancreatic, colorectal, melanoma, hepatocellular carcinoma, and others (22,23).Previous studies from our laboratory demonstrated that NDRG1 expression inhibited the TGF-b-induced epithelial-to-mesenchymal transition (EMT) (7).The ability of NDRG1 to inhibit the EMT occurs through (1) maintaining the cell membrane localization of E-cadherin and b-catenin that forms part of the adherens junction complex; (2) inhibiting the expression of the EMT marker, vimentin; and (3) blocking cell migration and invasion of prostate and colon cancer cells (7).
Protein kinase Ca (PKCa) is a member of the protein kinase C family of threonine-and serine-specific protein kinases that require calcium and diacylglycerol for activation (24).The PKCa isotype is ubiquitously expressed and is activated in response to a series of stimuli depending on the effector, and can result in PKCa translocation from the cytoplasm to specialized cellular compartments (24).Due to PKCa being sensitive to multiple stimuli (24), it has been implicated in apoptosis, proliferation, differentiation, inflammation, and motility (24).While PKCa plays a role in a range of pathways, there is increasing evidence that PKCa can regulate WNT/bcatenin signaling (20,(24)(25)(26).In fact, PKCa can associate with, and phosphorylate b-catenin at Ser33, Ser37, and Thr41, causing its degradation and the inhibition of WNT/b-catenin signaling (27).
Using several PC cell-types, the current study investigated the antagonistic effect of NDRG1 expression on WNT/b-catenin signaling and demonstrated the inhibition of this pathway by multi-modal mechanisms.These inhibitory activities of NDRG1 included: (1) the identification of a potential metabolon between PKCa, NDRG1, and b-catenin that could promote their catalytic activity.In fact, NDRG1 overexpression results in association of NDRG1 with PKCa and the stabilization of PKCa that phosphorylates b-catenin at Ser33, Ser37, and Thr41 leading to b-catenin degradation; (2) the decreased phosphorylation of b-catenin at Ser552 that suppresses its transcriptional activation (46); and (3) the downregulation of the nucleo-cytoplasmic shuttling protein p21-activated kinase 4 (PAK4), which prevents oncogenic nuclear translocation of b-catenin.Moreover, we report that innovative NDRG1inducing thiosemicarbazone drugs mimic genetic overexpression of NDRG1 to upregulate PKCa and downregulate b-catenin and its key downstream effector, cyclin D1.

NDRG1 overexpression in PC cells increases phosphorylation
of b-catenin at Ser33, Ser37, and Thr41 and downregulates the b-catenin nucleo-shuttling protein, PAK4 PC cells display aberrant WNT signaling (22,23), with previous studies demonstrating that NDRG1 overexpression antagonizes the WNT/b-catenin pathway in colon and prostate cancer cells (13).Since NDRG1 can also decrease nuclear b-catenin levels in PC cells (47), the mechanism of its activity in these tumor cells was important to examine.To investigate the effect of NDRG1 expression on WNT/b-catenin signaling, PANC-1 PC cells were stably transfected with an NDRG1 expression vector compared to their vector control (VC) transfected counterparts (Fig. 1A).Western blot analysis was initially performed to examine the effect of NDRG1 overexpression on the total cellular levels of key WNT/b-catenin pathway proteins.
The expression of NDRG1 was significantly (p < 0.001) upregulated in NDRG1 overexpressing cells versus VC cells (Fig. 1A).In VC cells, two endogenous bands for NDRG1 were detected at 41 kDa and 46 kDa (Fig. 1A), which are consistent with the previously reported NDRG1 isoforms that are the result of cleavage, processing, and/or differential phosphorylation (12,48,49).The upper (46 kDa) NDRG1 band is the key active isoform for metastasis suppression since (1) it is potently upregulated by bespoke NDRG1-inducing thiosemicarbazone drugs that inhibit tumor growth and metastasis (12,31,34); and (2) silencing the NDRG1 top band prevented it acting on downstream effectors (e.g., E-cadherin and b-catenin) that curtail the EMT and metastasis (7).In NDRG1 overexpressing cells, total NDRG1 levels were significantly (p < 0.001) greater than that observed in VC cells (Fig. 1A).In these latter cells, the flag-tagged transfected NDRG1 was detected at 46and 47-kDa, and the endogenous isoform was observed as a faint band at 41 kDa (Fig. 1A).The two isoforms of transfected NDRG1 could represent different post-translational modifications of the protein, as observed for endogenous NDRG1 (12,31,34).The densitometric analysis presented throughout this study represents the total of all NDRG1 bands detected in each cell-type.
Examining PANC-1 cells, western analysis demonstrated a marked and significant (p < 0.001) decrease in total b-catenin levels in the NDRG1 overexpressing cells versus the VC (Fig. 1A).This was clearly different from studies using prostate and colon cancer cells where NDRG1 expression caused no significant (p > 0.05) alteration in total b-catenin (13).The levels of b-catenin phosphorylation at Ser552, which is known for promoting b-catenin transcriptional activity via protein kinase B (AKT) (46), was also significantly (p < 0.001) decreased upon NDRG1 overexpression relative to the VC (Fig. 1A).
In Thr41) represents b-catenin in its most stable form (50).In contrast, NDRG1 overexpression resulted in a significant (p < 0.01) increase in b-catenin phosphorylated at Ser33, Ser37, and Thr41 (Fig. 1A), which causes destabilization of this protein and could, at least in part, explain the decreased total b-catenin levels.Therefore, this investigation demonstrates a novel mechanism of b-catenin regulation via NDRG1 in PC cells.
As AKT plays an important role in regulating the phosphorylation of b-catenin at Ser552 (46) and GSK-3b at Ser9 (51), its total protein levels and the activating phosphorylation of AKT at Ser473 (52) were also examined.As reported by our laboratory previously using PANC-1 cells (9), total AKT levels were not significantly (p > 0.05) affected by NDRG1 overexpression, while its phosphorylation at Ser473 was significantly (p < 0.001) decreased versus VC cells (Fig. 1A).This inactivation of AKT could explain the decreased phosphorylation of b-catenin at Ser552, but not the significant (p < 0.001) increase in GSK-3b phosphorylation at Ser9 after NDRG1 overexpression (described below; Fig. 1A).
The overexpression of NDRG1 also significantly (p < 0.01) downregulated PAK4 (Fig. 1A) which is involved in the stability and nuclear translocation of b-catenin (53,54).Therefore, the ability of NDRG1 overexpression to downregulate PAK4 expression, as well as total b-catenin levels, underlines the anti-oncogenic activity of NDRG1 overexpression in PANC-1 PC cells.

NDRG1 overexpression induces a GSK-3b-independent mechanism to decrease b-catenin expression that may be mediated by PKCa upregulation
The phosphorylation of b-catenin at Ser33, Ser37, and Thr41, which results in b-catenin degradation, is classically mediated by GSK-3b (55).Interestingly, in PANC-1 PC cells, the total levels of GSK-3b were markedly (p < 0.001) decreased upon NDRG1 overexpression versus the VC (Fig. 1A).Moreover, as described earlier, the inhibitory phosphorylation of GSK-3b at Ser9 (56) was significantly (p < 0.001) increased in NDRG1 overexpressing cells (Fig. 1A).In contrast, the activating phosphorylation of GSK-3b at Tyr216 (57) was significantly (p < 0.01) decreased in NDRG1 overexpressing cells (Fig. 1A).The decrease in total GSK-3b and its reduced activation after NDRG1 overexpression in PANC-1 PC cells demonstrates two significant points.First, GSK-3b was probably not responsible for the significant increase in bcatenin phosphorylation at Ser33, Ser37, and Thr41.Second, our study highlights a distinctive difference in the effect of NDRG1 overexpression in PC cells relative to our previous findings (13) using colon and prostate cancer cells where no significant (p > 0.05) change in total GSK-3b levels or its activation was observed.
However, similar to our previous investigation examining colon and prostate cancer cells (13), NDRG1 overexpression in PANC-1 cells significantly increased (p < 0.001) the expression of the GSK-3b-binding protein, frequently rearranged in advanced T-cell lymphoma (FRAT1), relative to VC cells (Fig. 1A).FRAT1 prevents GSK-3b association with the axin1-APC-CK1 destruction complex and the subsequent phosphorylation of b-catenin (58).This effect leads to the accumulation of non-phosphorylated b-catenin after NDRG1 expression in colon and prostate cancer cells (13).Since FRAT1 prevents phosphorylation of b-catenin by GSK-3b (58), these results further suggest the observed ability of NDRG1 overexpression in PC cells to increase b-catenin phosphorylation at Ser33, Ser37, and Thr41 (Fig. 1A) is not achieved by GSK-3b.This hypothesis is investigated in detail below.
While FRAT1 levels are increased in PANC-1 cells after NDRG1 overexpression, there was no increase in nonphosphorylated b-catenin (Fig. 1A).In fact, a pronounced and significant (p < 0.001) decrease in non-phosphorylated bcatenin was observed after NDRG1 overexpression versus the VC (Fig. 1A).These results indicate the NDRG1-mediated mechanism regulating b-catenin expression in PC cells is distinct from that observed in prostate and colon cancer cells (13).
Since GSK-3b did not appear responsible for the phosphorylation of b-catenin at Ser33, Ser37, and Thr41, it was hypothesized that PKCa could be responsible since it is known to phosphorylate b-catenin at these sites (27).The current studies demonstrated that NDRG1 overexpression significantly (p < 0.001) increased total PKCa levels versus the VC (Fig. 1A), suggesting a potential role of PKCa in b-catenin phosphorylation.The ability of NDRG1 overexpression to decrease total b-catenin expression and increase its phosphorylation at Ser 33, Ser37, and Thr41 should inhibit WNT signaling (59).Considering this, the expression of a key downstream effector of WNT signaling, namely cyclin D1, was significantly (p < 0.01) decreased after NDRG1 overexpression versus the VC (Fig. 1A).The upregulation of PKCa by the metastasis suppressor, NDRG1, is also of interest since PKCa acts as a tumor suppressor in other cancers because it promotes phosphatase 2A (PP2A)-family-dependent AKT inactivation (60).As such, the increase in PKCa observed after NDRG1 overexpression may lead to decreased p-AKT (Ser473) levels, which then results in decreased b-catenin phosphorylation at Ser552 (Fig. 1A).This hypothesis is examined further below in NDRG1 and PKCa silencing studies.
Considering the results above after NDRG1 overexpression in PANC-1 cells, another PC cell-type was also utilized, namely AsPC-1, with NDRG1 being silenced to examine its effect on WNT b-catenin signaling and PKCa expression (Fig. 1B).The AsPC-1 cells were used for silencing as our previous studies demonstrated that it has high endogenous NDRG1 levels enabling effective NDRG1 silencing (35).
To further corroborate that NDRG1 silencing increases GSK-3b levels and its activity, levels of the GSK-3b antagonist, FRAT1, were examined after silencing NDRG1 and were shown to be significantly (p < 0.01) decreased versus the siControl (Fig. 1B).Since FRAT1 inhibits GSK-3b binding to the destruction complex, which then prevents b-catenin phosphorylation (58), a decrease of FRAT1 in NDRG1 silenced cells should increase GSK-3b activity and increase phosphorylation of b-catenin at Ser33, Ser37, Thr41 (58).However, since a significant (p < 0.01) decrease in b-catenin phosphorylation at Ser33, Ser37, and Thr41 occurred after NDRG1 silencing (Fig. 1B), this again indicates a GSK-3b-independent mechanism for phosphorylating these later residues on bcatenin.
Considering the possible GSK-3b-independent mechanism involved in the phosphorylation of b-catenin at Ser33, Ser37, and Thr41, NDRG1 silencing in AsPC-1 cells demonstrated a significant (p < 0.001) decrease in PKCa versus the siControl (Fig. 1B).This observation was opposite to the PKCa upregulation observed after NDRG1 overexpression (Fig. 1A).The decrease in PKCa after NDRG1 silencing is consistent with the significant decrease in b-catenin phosphorylation at Ser33, Ser37, and Thr41 under these conditions (Fig. 1B).Such alterations that lead to increased b-catenin levels could promote WNT signaling (59).Consistent with this finding, the WNT downstream effector, cyclin D1, was significantly (p < 0.001) increased after NDRG1 silencing relative to VC cells (Fig. 1B).These results further support the role of PKCa in the GSK-3b-independent mechanism for mediating the phosphorylation of b-catenin at Ser33, Ser37, and Thr41 induced by NDRG1 overexpression.
PKCa silencing increases total b-catenin and decreases the destabilizing b-catenin phosphorylation at Ser33, Ser37, and

Thr41
To additionally investigate the functional role of PKCa in bcatenin phosphorylation, PKCa was silenced in PANC-1 and AsPC-1 cells using siRNA (Fig. 2, A and B).Silencing of PKCa resulted in a marked and significant (p < 0.001) decrease in PKCa levels in both PANC-1 (Fig. 2A) and AsPC-1 cells (Fig. 2B) versus their respective siControls.The levels of NDRG1 were not significantly (p > 0.05) affected by the silencing of PKCa in both cell-types (Fig. 2, A and B) compared to their siControls.This finding suggests that while NDRG1 overexpression acts as a positive regulator of PKCa expression (Fig. 1A), PKCa does not regulate NDRG1 levels.
As indicated previously, considerable cross-talk exists between AKT and PKCa signaling (60), with NDRG1 overexpression decreasing p-AKT (Ser473) levels and its downstream target p-b-catenin (Ser552; Fig. 1A).This latter effect may be mediated by the upregulation of PKCa after NDRG1 overexpression.This putative relationship was examined here by silencing PKCa, which significantly (p < 0.001) increased p-AKT (Ser473), but not total AKT levels versus the siControl in both PANC-1 and AsPC-1 PC cells (Fig. 2, A and  B).These findings are consistent with the idea of cross-talk between PKCa and AKT, suggesting that PKCa mediates a tumor suppressive role upon NDRG1 overexpression.
In contrast to the results with PANC-1 cells (Fig. 2A), when GSK-3b was examined in PKCa silenced AsPC-1 cells, the total protein levels, and inhibitory phosphorylation (Ser9) of GSK-3b were significantly (p < 0.001-0.01)increased versus the siControl (Fig. 2B).Previous studies examining the effect of PKCa knockout in mice led to similar results where total and p-Ser9 levels of GSK-3b increased relative to wildtype mice (63).The increased p-GSK-3b (Ser9) levels upon PKCa knockout were attributed to an increase in AKT (63), which catalyzes the inhibitory phosphorylation of GSK-3b at Ser9 (64).Therefore, the increase in p-GSK-3b (Ser9) in AsPC-1 cells may be due to the increase in AKT activation (Ser473) upon PKCa silencing (Fig. 2B).Interestingly, the activating GSK-3b phosphorylation at Tyr216 was not significantly (p > 0.05) altered in PKCa silenced AsPC-1 cells compared to the siControl (Fig. 2B).The different effects of PKCa silencing on GSK-3b observed in PANC-1 (Fig. 2A) and AsPC-1 cells (Fig. 2B) suggests the role of PKCa is celltype specific.Nonetheless, silencing PKCa increased total bcatenin expression in both cell-types and also resulted in the upregulation of cyclin D1 expression versus the siControl (Fig. 2, A and B), suggesting its role in regulating WNT signaling.
Overall, PKCa silencing in Figure 2, A and B is consistent with the conclusion that PKCa downregulates b-catenin by phosphorylating it at Ser33, Ser37, and Thr41, which causes bcatenin degradation.These silencing studies also confirm the inhibitory effect of NDRG1 overexpression on the WNT/bcatenin pathway (Fig. 1A) is caused, at least in part, by the upregulation of PKCa.

Confocal microscopy and co-immunoprecipitation (co-IP) demonstrate the association of NDRG1 and PKCa
The studies in Figures 1 and 2 support the hypothesis that NDRG1 inhibits the b-catenin pathway by increasing PKCa expression.As NDRG1 stabilizes and increases the expression of other proteins (e.g., MIG6, androgen receptor, O 6 -methylguanine-DNA methyltransferase, etc.) by promoting its association with these targets (17,65,66), confocal microscopy studies were initiated to examine the co-localization between NDRG1 and PKCa (Fig. 2C).In agreement with western analysis (Fig. 1A), these studies demonstrated a significant (p < 0.001) increase in NDRG1 and PKCa intensity in NDRG1 overexpressing cells relative to VC cells (Fig. 2C).Furthermore, NDRG1 and PKCa had a similar distribution in cells, being predominantly perinuclear (Fig. 2C).
Since confocal microscopy images are captured from a single lateral focal plane, proteins present in a similar location can be co-localized (67).There was a marked and significant (p < 0.001) increase of co-localization between NDRG1 and PKCa in NDRG1 overexpressing cells versus VC cells (Fig. 2C), suggesting an increased association between NDRG1 and PKCa.Similarly, a significant (p < 0.01) increase was observed in the Pearson correlation coefficient (r) upon NDRG1 overexpression versus the VC (from 0.18 ± 0.05-0.60 ± 0.11; Fig. 2C).
To further test the hypothesis that NDRG1 and PKCa associate, co-IP analysis was undertaken using PANC-1 cell model as the effects of NDRG1 are well characterized in this cell-type by our laboratory (9).The co-IP was performed using antibodies against both NDRG1 (Fig. 3A) or PKCa (Fig. 3B) to validate the interaction.An isotype control antibody (Isotype Con.Ab) was also utilized to assess the non-specific binding of immunoglobins to the protein of interest.Western analysis (input) of total cell lysates and co-IP using an NDRG1 antibody demonstrated significantly (p < 0.001) higher NDRG1 levels in NDRG1 overexpressing cells compared to VC cells (Fig. 3A).Examination of PKCa expression in NDRG1 overexpressing cells versus the VC also indicated significantly (p < 0.001) increased levels in the input and also after co-IP with an NDRG1 antibody (Fig. 3A).
Western analysis of the input and co-IP using a PKCa antibody demonstrated significantly (p < 0.001) higher PKCa levels in NDRG1 overexpressing cells (Fig. 3B).Furthermore, there was a significant (p < 0.001-0.01)increase of NDRG1 in the input and after co-IP with the PKCa antibody (Fig. 3B).For the co-IP investigations in Figure 3, A and B, the Isotype Con.Ab demonstrated very low non-specific binding and indicated the high specificity of the antibody used for IP of the target protein.
In summary, these results in Figure 3, A and B indicate a marked increase in the association of NDRG1 and PKCa in NDRG1 overexpressing cells versus the VC and support the evidence of this interaction from confocal microscopy in Figure 2C.

PKCa half-life is increased upon NDRG1 overexpression
It can be hypothesized from the studies in Figures 2C and 3, A and B that NDRG1 associates with PKCa, stabilizes PKCa, and potentially increases its half-life, as reported for the association of NDRG1 with other proteins (17,65,66).To examine this, the half-life of PKCa in PANC-1 NDRG1overexpressing cells was compared to that in VC cells using the protein synthesis inhibitor, cycloheximide (CHX; Fig. 3C).Western blot analysis clearly demonstrated the higher NDRG1 levels in the NDRG1 overexpressing clone versus the VC.
However, upon incubation with CHX (15 mg/ml), there was no significant (p > 0.05) change in NDRG1 levels for incubations up to 14 h in both cell-types relative to the 0 h control (Fig. 3C).In marked contrast, examining VC cells a pronounced decrease in PKCa expression was observed after an incubation of 2 h versus the 0 h control.On the other hand, examining NDRG1 overexpressing cells, a decrease in PKCa levels relative to the 0 h control was only apparent after a 10 h incubation (Fig. 3C).Calculation of the half-life of PKCa from these data demonstrated that it was significantly (p < 0.001) increased from 1.5 ± 0.8 h (3) in VC cells to 11.0 ± 2.5 h (3) in NDRG1-overexpressing cells, respectively (Fig. 3C).In conclusion, the protein half-life of PKCa is markedly increased upon NDRG1 expression in PANC-1 cells, indicating PKCa stabilization.
NDRG1 overexpression results in a significant association between PKCa and b-catenin and a decrease in b-catenin expression Apart from the association of NDRG1 with PKCa demonstrated previously (Figs.2C and 3, A and B), PKCa has been reported to directly bind to the armadillo repeat sequences of b-catenin (27), Together, these observations suggest a complex interactome of proteins that required further investigation that was initially performed using confocal microscopy (Fig. 4,  A-F).
Studies were first designed to investigate the association between PKCa with b-catenin using NDRG1 overexpressing PANC-1 cells versus their VC counterparts.In these experiments, cells were incubated for 24 h in the absence (Fig. 4A) or presence (Fig. 4B) of the WNT ligand, WNT3a (100 ng/ml), which stimulates nuclear translocation of b-catenin (68).In the absence of WNT3a in VC cells, b-catenin demonstrated predominantly cytoplasmic expression, while little PKCa was apparent (Fig. 4A).Examining PANC-1 cells with NDRG1 overexpression in the absence of WNT3a, there was a NDRG1 inhibits WNT/b-catenin signaling by stabilizing PKCa significant (p < 0.01) decrease in total cellular b-catenin levels (Fig. 4C) and a pronounced and significant (p < 0.001) increase in PKCa expression (Fig. 4D).These latter results confirmed the findings of western analysis under analogous conditions (Fig. 1A).
In NDRG1 overexpression versus VC cells (Fig. 4F).The colocalization of PKCa (red) with DAPI (blue) resulting in purple nuclei (Fig. 4A) indicated this protein demonstrated some nuclear translocation after NDRG1 overexpression.Previous studies under several conditions have also demonstrated PKCa translocation to the nucleus (69,70).
As expected from its role in activating WNT signaling (68), upon incubating VC cells with WNT3a, there was a significant (p < 0.001) increase in total (Fig. 4, B, C, and E) and nuclear bcatenin expression (Fig. 4E) versus VC cells without WNT3a.The addition of WNT3a also resulted in a slight increase (p > 0.05) in PKCa expression in VC cells relative to VC cells incubated without WNT3a (Fig. 4D).Some limited nuclear colocalization of PKCa, b-catenin, and DAPI was evident (white fluorescence) in VC cells treated with WNT3a relative to VC cells without WNT3a (Fig. 4B).Only very sparse colocalization (yellow) of b-catenin and PKCa was demonstrated in VC cells after incubation with WNT3a, but this was not significantly (p > 0.05) different to VC cells without WNT3a (Fig. 4, B and F).
After the addition of WNT3a to NDRG1 overexpressing cells there was a significant (p < 0.001) decrease in total (Fig. 4, B and C) and nuclear b-catenin levels (Fig. 4, B and E) versus the VC incubated with WNT3a.In the presence WNT3a, there was also a significant (p < 0.001) increase in PKCa in NDRG1 overexpressing cells relative to VC cells (Fig. 4, A and D).Due to the decrease in nuclear b-catenin levels after NDRG1 overexpression in the presence of WNT3a, no marked colocalization (white) was observed between DAPI (blue), PKCa (red), and b-catenin (green; Fig. 4, B and E).However, in the presence of WNT3a and after NDRG1 overexpression, there was a pronounced and significant (p < 0.001) increase in co-localization (yellow) between PKCa and b-catenin relative to VC cells (Fig. 4, B and F).
In summary, confocal microscopy in Figure 4  In addition to the interaction between PKCa and b-catenin (Fig. 4), NDRG1 has also been described as associated with bcatenin in esophageal squamous carcinoma cells (71).To examine if this also occurs in PC cells, the cellular distribution and association between b-catenin (green) and NDRG1 (red) was examined by confocal microscopy in the absence and presence of WNT3a in PANC-1 VC and NDRG1 overexpressing cells (Fig. 5, A-F).Investigating VC cells in the absence of WNT3a, there were appreciable total b-catenin levels (Fig. 5, A and C) and low NDRG1 expression (Fig. 5, A  and D) that led to very limited cytoplasmic co-localization (yellow; Fig. 5, A and F).Overexpression of NDRG1 in the absence of WNT3a resulted in significantly (p < 0.01) decreased total cellular b-catenin expression versus the VC (Fig. 5, A and C).In the absence of WNT3a, NDRG1 overexpression led to a significant (p < 0.001) increase in cytoplasmic and membrane co-localization of NDRG1 and bcatenin relative to VC cells (Fig. 5, A and F).
The addition of WNT3a to VC cells, significantly (p < 0.001) increased total (Fig. 5 Notably, comparing NDRG1 overexpressing and VC cells in the presence of WNT3a, despite the significant (p < 0.001) decrease in total cellular b-catenin levels (Fig. 5, B and C), the co-localization of NDRG1 and b-catenin was significantly (p < 0.001) enhanced and was predominantly at the plasma membrane (Fig. 5B).This observation can be explained by our previous studies demonstrating the ability of NDRG1 to stabilize b-catenin at the plasma membrane as part of the adherens junction, which maintains cellular adhesion and inhibits the EMT (7).However, the co-localization between b-catenin and NDRG1 was significantly (p < 0.001) less pronounced in the presence of WNT3a than in its absence (Fig. 5F).
Considering the association between PKCa and NDRG1 (Figs. 2C and 3, A and B), PKCa and b-catenin (Fig. 4, A, B, and F), and NDRG1 and b-catenin (Fig. 5, A, B, and F), it was considered if all three proteins may associate (Fig. 6, A-H).In fact, the triple co-localization (white) of PKCa (blue), NDRG1 (red), and b-catenin (green) was significantly (p < 0.001) increased after NDRG1 overexpression, in the absence and especially the presence of WNT3a (Fig. 6H).These studies suggest an association between these three proteins that occurred primarily in the perinuclear region of the cell.NDRG1 inhibits WNT/b-catenin signaling by stabilizing PKCa N1 cells versus VC cells (Fig. 7C).For all co-IP investigations in Figure 7, A-C, the Isotype Con.Ab demonstrated very low non-specific binding and indicated the high specificity of the antibody used for IP of the target protein.

Overall, confocal microscopy in
Collectively, these data in Figure 7 together with the confocal microscopy results in Figures 4-6 are evidence of an association between NDRG1 and b-catenin and also PKCa and b-catenin.The 3 latter proteins potentially form a metabolon that could be important for facilitating their catalytic activity.

Pharmacological induction of NDRG1 mimics genetic overexpression of this protein to upregulate PKCa and decrease b-catenin levels
Metastasis is the major killer in cancer (72,73) and NDRG1 is a potent metastasis suppressor in multiple cancers (1)(2)(3)(4)(5)(6)(7)(8)(9)(10).Understanding the anti-oncogenic mechanism of action of NDRG1 is important regarding the development of innovative drugs in our laboratory that induce its expression (4,44).Anti-cancer agents of the di-2-pyridylketone thiosemicarbazone class e.g., di-2- and PKCa associate upon NDRG1 overexpression.PANC-1 VC and NDRG1 cells were incubated with a control medium at 37 C for 24 h and then lysed using IP lysis buffer and 400 mg of whole cell lysate incubated overnight at 4 C with either: (A) NDRG1, (B) PKCa, or (C) b-catenin antibodies or a rabbit isotype control antibody (Isotope Con.Ab).Protein A/G bound to magnetic beads was then added to cell lysates and incubated for 1.5 h/4 C. Samples were placed on a magnet to isolate antibody/protein complexes, which were then subjected to western blotting.Densitometry is presented as the mean ± SD (n = 3).Statistical significance is denoted as ***p < 0.001 comparing PANC-1 NDRG1 versus VC cells.
Considering the ability of NDRG1 overexpression to upregulate PKCa expression and inhibit WNT signaling (Fig. 1A), it was important to understand if analogous activity could be identified after treatment of PC cells with the NDRG1-inducing thiosemicarbazones (Fig. 8).In these studies, PANC-1 cells were incubated for 24 h/37 C with either a control medium or this medium containing the NDRG1-inducing thiosemicarbazones, namely Dp44mT (5 mM), DpC (5 mM), or PPP44mT (5 mM).A number of controls were also utilized, including the negative control compound for the thiosemicarbazones, namely 2benzoylpyridine-2-methylthiosemicarbazone (Bp2mT; 5 mM).
This agent has been specifically designed by our laboratory not to bind iron or other metals (76), and thus, cannot upregulate NDRG1 and does not possess biological activity (17-19, 33, 35, 41, 74).A positive control was also included, namely desferrioxamine (DFO; 100 mM), which is a well-known iron-binding ligand that is clinically used for the treatment of iron overload disease (77,78) that upregulates NDRG1 (33,74).The concentration of DFO used was higher herein due to its membrane permeability being limited (79) and considerably lower than the thiosemicarbazones (37).
The phosphorylation of b-catenin at Ser33, Ser37, and Thr41 was significantly (p < 0.001) increased relative to the control by Dp44mT, DpC and PPP44mT.Generally, the positive control, DFO, was less effective than the thiosemicarbazones at regulating b-catenin and its phosphorylation, and this may relate to its poor cellular permeability (37,79).Furthermore, PAK4, which is involved in the stability and nucleocytoplasmic translocation of b-catenin (53,54), was significantly (p < 0.001-0.05)downregulated by DFO, Dp44mT, DpC, and PPP44mT, as demonstrated for genetic NDRG1 overexpression (Fig. 1A).Overall, the pharmacological induction of NDRG1 by the thiosemicarbazones resulted in similar effects on the regulation of b-catenin as genetic NDRG1 overexpression.
In agreement with the upregulation of NDRG1 and PKCa and downregulation of b-catenin expression by Dp44mT, DpC, and PPP44mT that could suppress WNT signaling, the downstream effector of this pathway, cyclin D1, was significantly (p < 0.001) downregulated by these agents versus the control (Fig. 8).Overall, the NDRG1-inducing agents mimicked genetic NDRG1 overexpression resulting in the upregulation of PKCa and multiple alterations in the expression of WNT pathway proteins that inhibit downstream WNT signaling.

Discussion
PC is a highly belligerent and aggressive disease (28, 29, 80), where existing treatments are poorly effective.As such, understanding pro-oncogenic molecular pathways, for example, WNT signaling that plays a role in the pathogenesis of PC (30) and the identification of new anti-oncogenic targets, is an important research aim.Aberrant WNT/b-catenin signaling is well known in PC (22), with the metastasis suppressor, NDRG1, antagonizing this and other oncogenic signaling pathways in multiple cancer cell-types (7,13), including PC cells and tumors (35,47).However, the mechanisms involved remain incompletely characterized and were the subject of this investigation.
Phosphorylation of b-catenin at Ser33, Ser37, and Thr4 is a major control point in the WNT pathway that is classically mediated by GSK-3b, which results in proteasome degradation of b-catenin (57).The current work demonstrated that the critical phosphorylation of b-catenin at Ser33, Ser37, and Thr41 in PC cells was independent of GSK-3b after NDRG1 overexpression.Considering a previous report that PKCa could phosphorylate b-catenin at these sites (27), PKCa was then hypothesized to be the kinase responsible, with this conclusion being supported by NDRG1 overexpression and silencing studies in two PC cell-types.These results suggested that after NDRG1 overexpression, PKCa phosphorylated bcatenin at Ser33, Ser37, and Thr41 leading to downregulation of b-catenin.Considering the mechanism involved, NDRG1 was shown to associate with PKCa and stabilize it to facilitate its catalytic function.
Previous investigations have reported a direct interaction of NDRG1 with b-catenin (71), with PKCa interacting with the armadillo repeat sequences of b-catenin (27).Our studies have demonstrated that PKCa associates with b-catenin, and additionally for the first time, NDRG1.An interaction of NDRG1 and b-catenin was also evident, suggesting the formation of a metabolon, with this study being the first to indicate all three proteins (NDRG1, PKCa, and b-catenin) associated.As observed for many other molecules (17,(81)(82)(83)(84)(85), the formation of a metabolon can promote the activity of these proteins and ensure effective catalytic processing.Generally, it is known that proteins can associate and interact with one another on a genome-wide scale (86) to maintain stability (87), and can facilitate biological signaling even after the failure of some of their components (88).
As we show herein, NDRG1 associates with PKCa upon NDRG1 expression resulting in PKCa stabilization (i.e., increased PKCa half-life).We have also demonstrated that NDRG1 overexpression increases phosphorylation of PKCa/b II (Thr638/641; Fig. S1A), which is important for maintaining PKCa catalytic competence (89).However, there was no change in the ratio of p-PKCa/b to total PKCa (Fig. S1A), indicating the increase in p-PKCa/b is due to the elevation in PKCa protein levels.Silencing of NDRG1 had the opposite effect to NDRG1 overexpression, decreasing PKCa phosphorylation and total levels of PKCa, while having no effect on their ratio (Fig. S1B).Nonetheless, the current study demonstrates the upregulation of PKCa promotes association with bcatenin, leading to its phosphorylation at Ser33, Ser37, and Thr41, resulting in the downregulation of b-catenin that is anti-oncogenic (Fig. 9).
Our previous investigations examining the effect of NDRG1 overexpression on WNT signaling in prostate and colon cancer cells demonstrated a distinctly different mechanism (13) to that reported herein.In fact, NDRG1 expression increased total and non-phosphorylated (Ser33, Ser37, and Thr41) b-catenin, while there was a decrease in the phosphorylation of b-catenin at Ser33, Ser37, and Thr41 (13).The mechanism of inhibiting b-catenin phosphorylation in these latter cell-types was not mediated by significant alterations in GSK-3b total levels nor its activation but involved NDRG1-mediated upregulation of FRAT1, and downregulation of PAK4 (13).In this prior work, the effects in prostate and colon cancer cells were consistent with the role of FRAT1 in preventing the association of GSK-3b with the destruction complex, while the downregulation of PAK4 inhibited nuclear translocation of b-catenin (13).These effects of NDRG1 were also observed in this investigation that accompanied PKCa stabilization (Fig. 9).Thus, NDRG1 demonstrates multi-modal mechanisms in suppressing WNT/b-catenin signaling in PC cells.Yet other mechanisms that mediate the activity of NDRG1 have been reported examining Huh7 and HepG2 hepatocellular carcinoma cells, where direct binding of NDRG1 to GSK-3b prevented its binding to b-catenin (90).This later effect of NDRG1 inhibited GSK-3b-mediated phosphorylation of b-catenin at Ser33, Ser37, and Thr41 (90).
Both the present investigation examining PC cells and our previous studies using prostate cancer and colon cancer cells (13) also demonstrated that NDRG1 expression inhibited bcatenin phosphorylation at Ser552 promoting its transcriptional activity (46).It has been additionally shown that activated AKT can phosphorylate GSK-3b at Ser9 (64).However, after NDRG1 overexpression, AKT activation is suppressed, with decreased p-AKT (Ser473) levels being observed.As such, the increased phosphorylation of GSK-3b (Ser9) after NDRG1 expression is consistent with an AKT-independent mechanism.There is also considerable crosstalk between AKT and PKCa in endometrial cancer, with AKT activation being negatively regulated by PKCa that had an anti-oncogenic effect (64).Similarly, in PC cells, PKCa silencing increased p-AKT (Ser473) and p-b-catenin (Ser552) levels that were consistent with the increase of PKCa after NDRG1 overexpression decreasing p-AKT (Ser473) and p-b-catenin (Ser552; Fig. 9).
Using neural cells (91) and platelets (63), studies implementing chemical inhibitors provided evidence that PKCa phosphorylates GSK-3b at Ser9 inhibiting its activity.This observation suggested the NDRG1-mediated upregulation of PKCa observed in PANC-1 cells after NDRG1 overexpression could be responsible for the increase in GSK-3b phosphorylation at Ser9 (Fig. 1A).However, PKCa silencing had no effect on GSK-3b Ser9 phosphorylation in PANC-1 cells, or paradoxically increased it in AsPC-1 cells, suggesting PKCa was not responsible for GSK-3b phosphorylation at Ser9 under these conditions.
PC remains a belligerent, leading cause of cancer-related death that has unfortunately doubled worldwide in the past 25 years (92).Current chemotherapies are woefully suboptimal, leading to an overall 5-year relative survival rate of approximately 10% (93).New therapeutics with a different mechanism of action are desperately needed.Metastasis is the major killer in cancer leading to 90% of deaths (72,73).As such, there is an urgent need to identify novel targets and use that knowledge to specifically design and develop bespoke anti-metastatic drugs, as none exist in the clinics (94,95).The metastasis suppressor, NDRG1, has recently become recognized as an important pharmacological target (96,97).Our previous studies designed and synthesized novel thiosemicarbazone anti-cancer drugs that induce NDRG1 expression and potently inhibit primary tumor growth in vivo in a variety of cancers including several PC models (16,34,(37)(38)(39)(40)(41)(42)98).
The high potential of the thiosemicarbazones led to a Phase I clinical trial of our thiosemicarbazone anti-cancer drug, DpC (99,100), and the subsequent synthesis of a new generation of analogs, including PPP44mT, that are patented and undergoing development (33,74,101).The mechanism of action of NDRG1 is mediated through the inhibition of multiple oncogenic pathways, including WNT signaling (16,17,47,102).The ability of these thiosemicarbazones to upregulate NDRG1 expression has been demonstrated by our laboratory and others to (1) inhibit oncogenic WNT signaling; (2) decrease the nuclear translocation of b-catenin; and (3) inhibit the initial first step of metastasis, the epithelial-mesenchymal transition (7).Of note, the marked and selectively antiproliferative and/or anti-metastatic activity of Dp44mT, DpC, and PPP44mT have been well characterized in vitro and/ or in vivo (16,33,34,38,39,41,74).
The current investigation dissects novel molecular mechanisms involved in the inhibition of WNT signaling with our studies demonstrating that Dp44mT, DpC, and PPP44mT mimic genetically overexpressed NDRG1 by upregulating PKCa, downregulating b-catenin and PAK4, inhibiting GSK-3b expression and activation, and suppressing the expression of a key WNT effector, cyclin D1.Targeting WNT signaling has been described as a potential therapeutic strategy for PC (30), with the novel molecular mechanisms dissected herein not being observed in other clinically used pharmacopoeia.As such, the development of these thiosemicarbazones as effective anti-cancer agents warrants further vigorous investigation.
In conclusion, for the first time, the current investigation reports a novel mechanism of NDRG1-mediated antagonism of WNT signaling in PC cells facilitated by a novel metabolon involving the association of NDRG1, PKCa, and b-catenin that may effectively promote the activity of these proteins (Fig. 9).In fact, an association of NDRG1 with PKCa was demonstrated that may lead to the observed stabilization of PKCa and its increased protein levels.The upregulation of PKCa mediated the destabilizing phosphorylation of b-catenin at Ser33, Ser37, and Thr41, and a marked decrease in its expression (Fig. 9).This effect of NDRG1 expression was accompanied by the downregulation of PAK4 that inhibits b-catenin nuclear translocation.Finally, NDRG1 overexpression upregulates PKCa that suppresses AKT phosphorylation at Ser473, and the phosphorylation of b-catenin at Ser552 that promotes its transcriptional activity (Fig. 9).Dissection of these mechanisms is important in understanding the potent pharmacological inhibition of PC growth by innovative pharmacopeia of the thiosemicarbazone class that are under development as anti-cancer drugs (16,18,34).
PANC-1 cells were grown using Dulbecco's Modified Eagle Medium (DMEM) containing 10% (v/v) fetal bovine serum (FBS), 1% non-essential amino acids (Cat.#:M7145, Sigma Aldrich), 1% streptomycin/penicillin/glutamine (Cat.#:G1146, Sigma Aldrich) and 1% sodium pyruvate (Cat.#:S8636, Sigma Aldrich).PANC-1 VC and NDRG1 cells were also grown and maintained in the same medium with 0.4 mg/ml G418 (Geneticin; Thermo Fisher Scientific) to select for cells transfected with the plasmid.AsPC-1 pancreatic cancer cells, SH-SY5Y and KELLY neuroblastoma cells were grown in Roswell Park Memorial Institute (RPMI) media, which included the same supplements listed above for DMEM.T47D breast cancer cells were grown in DMEM media with the supplements above.SK-Mel-28 melanoma cells, MCF-7 breast cancer cells were grown in Eagle's modified minimum essential media (MEM), with the supplements above.All cells were grown in an incubator at 37 C with an atmosphere of 5% CO 2 /95% air.In experiments studying the effect of WNT3a ligand, human WNT3a (Cat#: H17001, Sigma Aldrich); was reconstituted to 100 mg/ml in PBS containing 0.1% endotoxinfree human serum albumin.PANC-1 VC and NDRG1 cells were incubated with or without WNT3a (100 ng/ml)-containing DMEM media for 24 h/37 C.

Protein extraction
Following the removal of the cells from the culture plates, the suspensions were disrupted on ice using a sonicator 150 (Branson, MO), followed by centrifugation at 13,200 rpm/ 40 min/4 C using a Fresco 17 centrifuge (Thermo Fisher Scientific).Subsequently, the supernatant was separated from the pellet, and the protein concentration determined using the bicinchoninic acid (BCA) protein assay kit (Thermo Fisher Scientific) using a UV-Vis spectrophotometer (Shimadzu).

SDS-PAGE and Western blot analysis
Protein lysates were prepared after the addition of b-mercaptoethanol (Sigma-Aldrich) and then heated for 5 min/95 C on a heating block (103).SDS-PAGE (8, 10, or 12% gels) was then performed and the separated proteins were transferred for 16 h onto a PVDF membrane (0.45 mM pore size; Millipore) at 30 V/4 C. Once the transfer was complete, the membrane was soaked in 100% methanol for 30 s and dried at 37 C/2 h.
Membranes were blocked in 5% skim milk or bovine serum albumin (BSA; Cat.#: A9418 Sigma-Aldrich) solution in TBS-T at room temperature for 1.5 h.Primary antibodies were diluted in 5% skim milk or BSA in TBS-T and incubated with the appropriate membranes overnight at 4 C.The membranes were then incubated with secondary antibody diluted in 5% skim milk at room temperature for 1 h, followed by washing in TBS-T (3 times/5 min).A list of primary and secondary antibodies including catalogue numbers are provided in Table S1.The specificity of each antibody was validated by the suppliers and supported by the assessment of the molecular weight of each protein detected (using molecular weight markers).
The antibody-antigen complex was detected after incubation with Western HRP substrate (Cat no.WBLUF0500: Millipore) for 1 min.The signal produced was detected and imaged with a Sapphire Biomolecular Imager (Azure Biosystems, CA).Signals were normalized to the total protein loaded to each lane (80 mg/lane), with equal loading being determined by reference to the house keeping protein, b-actin (Table S1).For every Western blot performed, b-actin was also probed to ensure appropriate normalization of protein loading.

Co-immunoprecipitation (co-IP)
Protein extraction from cells was performed using immunoprecipitation lysis buffer (Pierce IP Lysis Buffer, Thermo Fisher Scientific).Standard methods previously used in our laboratory were utilized for co-immunoprecipitation (co-IP) (66).Lysate protein concentration was determined using the BCA protein assay described above.Then, 400 mg of protein was incubated overnight with either the primary antibody specific to the target protein (Table S1) or a respective isotype control antibody (Isotype Con.Ab; Cell Signaling Technology; Table S1) used to assess the non-specific binding of immunoglobulins to the protein of interest.
Magnetic bead solution (30 mg/ml; Pierce Protein A/G Magnetic Beads; Thermo Fisher Scientific) was blocked using 5% BSA/PBS solution overnight and added to the antibody: lysate solution and incubated for 1.5 h/4 C. The samples containing the magnetic beads and antibody: lysate solution were then placed on a magnet and the supernatant was discarded.The precipitate was washed 5 times with ice-cold PBS NDRG1 inhibits WNT/b-catenin signaling by stabilizing PKCa to remove non-specifically bound proteins on the magnetic beads.Then, the magnetic beads were suspended in 5× loading dye containing b-mercaptoethanol (Sigma-Aldrich) solution, and the bound proteins denatured by boiling at 5 min/95 C.This solution was used for SDS-PAGE and western blotting.
For comparison, input samples containing 80 mg of protein were run simultaneously using SDS-PAGE implementing the western blotting protocol above.

Cycloheximide chase studies
The half-lives of NDRG1 and PKCa were evaluated using a cycloheximide (CHX) chase assay implementing previously established methods (17).PANC-1 VC and NDRG1 cells were seeded and incubated overnight at 37 C and the cells were preincubated for 1 h/37 C with the protein synthesis inhibitor, CHX (15 mg/ml; Sigma-Aldrich).A chase was then performed for 0, 2, 4, 8, 10, 12, and 14 h/37 C in the presence of CHX (15 mg/ml).Subsequently, the cells were lysed, and protein extracted for SDS-PAGE and Western blotting.

Confocal microscopy
Initially, PANC-1 VC and NDRG1 cells (400,000 cells/ condition) were seeded on sterile coverslips and incubated for 24 h/37 C in media supplemented with serum or WNT3a ligand (100 ng/ml).Fixation was then performed by implementing a 10 min incubation with 4% (w/v) paraformaldehyde/ PBS/20 C, followed by cell permeabilization using 0.1% Triton X-100/PBS using an incubation for 10 min/20 C. Following permeabilization, fixed cells were washed again, and the coverslips were blocked for 1 h with 5% BSA/0.3M glycine in PBS/20 C.Then, primary antibodies were diluted in 1% BSA/ PBS, added to each well, and incubated overnight at 4 C on an orbital shaker at 200 rpm.
After overnight incubation with the primary antibodies, the wells were washed three times with 1% BSA/PBS.Secondary antibody solutions were prepared to the desired concentration in 1% BSA/PBS and added to all wells, followed by incubation at room temperature for 1 h on an orbital shaker at 200 rpm.Coverslips were subsequently washed and mounted onto a slide using ProLong Gold DAPI mounting solution (Cat.# P36935; Invitrogen).The slides were then left to dry in the dark at room temperature for a minimum of 6 h.An Olympus FV3000RS confocal microscope (Evident Scientific) was used with a 60× and 100× objective to visualize cells.The images of visualized cells were then examined using Olympus Fluoview software, and in some studies, images were processed for deconvolution analysis with CellSens software (Olympus) to improve contrast and image resolution.Fluorescence intensities and co-localization analysis were quantified using ImageJ software (NIHD).The Pearson correlation coefficient (r) was calculated using the Image J plugin, JACoP.All primary and secondary antibodies used for confocal microscopy are listed in Table S1.
In order to maintain uniformity samples for the accurate assessment of colocalization, the following measures were implemented during sample preparation and the image acquisition process; (1) A consistent number of cells were seeded on each coverslip per sample to minimize variation in sample thickness and to ensure the presence of only a monolayer of cells; (2) images of each channels per sample were acquired at the same XYZ plane (relative to other channels); (3) within each experiment all microscope acquisition parameters (e.g., pinhole aperture settings) were kept constant.
The primary and secondary antibodies for immunostaining in the confocal analysis were validated by the manufacturers to be suitable for immunofluorescence imaging.In addition, the following measures were employed to further validate the antibodies: (1) Utilizing negative controls, such as omitting the primary antibodies (2) employing isotype controls (i.e., implementing isotype control antibodies); with matching concentration to the primary antibody of interest; and (3) adding positive controls (i.e., validated cell-types that possess the molecule of interest overexpressed and/or silenced).

Statistical analysis
Results for all experiments were generated from three independent experiments and presented as mean ± standard deviation.Data were normally distributed around the mean with a two-tailed Student's paired t test being used for statistical analysis implementing GraphPad Prism 9.2.0 software.Data were considered statistically significant when p < 0.05.Conflict of interest-The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

BFigure 1 .
Figure 1.NDRG1overexpression in PC cells increases phosphorylation of b-catenin at Ser33, Ser37, and Thr41 and downregulates the b-catenin nucleo-shuttling protein, PAK4.A and B, NDRG1 overexpression decreases activation of WNT/b-catenin signaling in PANC-1 PC cells, while silencing NDRG1 in AsPC-1 PC cells results in an opposite effect.PANC-1 vector control (VC) and PANC-1 NDRG1 overexpressing cells (NDRG1) were incubated with a control medium at 37 C for 24 h/37 C. Total cell protein lysate was then subjected to SDS-PAGE and Western blot analysis.b-actin was used as a protein loading control.B, AsPC-1 PC cells were incubated in control medium for 24 h/37 C. The cells were then transiently transfected with NDRG1 siRNA or nontargeting negative control siRNA (siControl) for 24 h/37 C. Total cell lysate was subsequently subjected to SDS-PAGE and Western blot analysis as in (A).Results are presented as the mean ± SD (n = 3).Statistical significance is denoted as **p < 0.01 and ***p < 0.001 comparing VC cells versus NDRG1 overexpressing cells.

Figure 2 .
Figure 2. PKCa silencing increases total b-catenin and decreases the destabilizing b-catenin phosphorylation at Ser33, Ser37, and Thr41, via possible association with NDRG1.A, PKCa silencing in PANC-1 cells results in an increase in b-catenin levels while having no significant effect on GSK-3b expression.PANC-1 PC cells were incubated with control medium for 24 h/37 C and then transiently transfected with PKCa siRNA or non-targeting negative control siRNA (siControl) for 24 h/37 C. Total cell protein lysate then underwent SDS-PAGE and analyzed via western blotting.b-actin was used as a protein-loading control.The blot is representative of three experiments, while the densitometry is mean ± SD (n = 3).B, PKCa silencing in AsPC-1 cells results in an increase of total b-catenin levels, while increasing the inhibitory Ser9 phosphorylation of GSK-3b.AsPC-1 PC cells were incubated with control medium for 24 h/37 C and then transiently transfected with PKCa siRNA or non-targeting negative control siRNA (siControl) for 24 h/37 C. Total cell protein lysate underwent SDS-PAGE and was analyzed via western blotting as in (A).C, confocal microscopy demonstrates that NDRG1 overexpression in PANC-1 cells increases the association between NDRG1 and PKCa.VC and NDRG1 overexpressing PANC-1 cells were examined for NDRG1 and PKCa expression and localization using confocal microscopy (60× objective) to demonstrate NDRG1 (green) and PKCa (red) staining.Yellow arrows show co-localization between NDRG1 and PKCa.The nucleus was stained by DAPI (blue).Images are representative of three experiments with pixel intensities and co-localization of NDRG1 and PKCa being quantitated with ImageJ using 24 cells.The Pearson correlation coefficient (r) was calculated using the Image J plugin, JACoP.Quantitation of the results represents the mean ± SD (n = 3).The scale bar = 15 mm except for the close-up image, where it is 3 mm.Statistical significance in (A-C) is denoted as *p < 0.05, **p < 0.01 and ***p < 0.001 comparing siControl with siPKCa, or VC and NDRG1 overexpression.

Figure 3 .
Figure 3. Co-immunoprecipitation demonstrates the association of NDRG1 and PKCa, extending the half-life of PKCa.A and B, association between NDRG1 and PKCa significantly increases in NDRG1 overexpressing cells as does (C) PKCa half-life.PANC-1 VC and NDRG1 cells were incubated with control medium at 37 C/24 h and then lysed using IP lysis buffer and 400 mg of whole cell lysate incubated overnight at 4 C with either: (A) NDRG1 or (B) PKCa antibodies or an isotype control antibody (Isotype Con.Ab).Protein A/G bound to magnetic beads was then added to cell lysates and incubated for 1.5 h/4 C. Samples were placed on a magnet to isolate antibody/protein complexes, which were then subjected to western blotting.C, PANC-1 VC and NDRG1 cells were preincubated with the protein synthesis inhibitor, cycloheximide (CHX; 15 mg/ml), for 1 h/37 C and then chased for 2 to 14 h/37 C with CHX (15 mg/ ml), harvested, and western analysis performed.Densitometry is presented as the mean ± SD (n = 3).Statistical significance is denoted as **p < 0.01 and ***p < 0.001 comparing NDRG1 overexpressing cells versus VC cells.

Figure 4 .
Figure 4. NDRG1 overexpression results in a significant association between PKCa and b-catenin and a decrease in b-catenin expression in the presence or absence of WNT3a.A-F, NDRG1 overexpression in PANC-1 cells significantly increases co-localization between b-catenin and PKCa in the presence and absence of WNT3a.A and B, VC and NDRG1 overexpressing PANC-1 cells were incubated for 24 h/37 C in the presence and absence of WNT3a (100 ng/ml).The cells were then examined for b-catenin (green) and PKCa (red) expression and co-localization (yellow) using confocal immunofluorescence microscopy.Cell nuclei were stained by DAPI (blue).Images are representative of three experiments and the quantitative analysis of intensities presented for: (C) b-catenin; (D) PKCa; (E) nuclear b-catenin; and (F) co-localization between PKCa and b-catenin.Studies were performed using a 60× objective at the same acquisition setting with Olympus Fluoview FV3000 software.ImageJ software was used for the analysis and the Pearson correlation coefficient (r) was calculated using the Image J plugin, JACoP.Quantitative analyses are presented as the mean ± SD (n = 3).Analysis of pixel intensity and co-localization were performed using 24 cells.The scale bar is 15 mm except for the close-up images, where it is 6 mm.Statistical significance is denoted as *p < 0.05, **p < 0.01 and ***p < 0.001 comparing NDRG1 overexpressing PANC-1 cells in the presence or absence of WNT3a to VC cells in the absence of WNT3a; or ### p < 0.001 comparing NDRG1 overexpressing PANC-1 cells to VC cells in the presence of WNT3a.
demonstrated that NDRG1 overexpression increased PKCa expression in the presence and absence of WNT3a and resulted in colocalization between PKCa and b-catenin.Further, NDRG1 overexpression demonstrated anti-oncogenic activity resulting in decreased total and nuclear b-catenin levels relative to VC cells in the presence of WNT3a.NDRG1 co-localizes with b-catenin upon NDRG1 overexpression and decreases total b-catenin levels and its nuclear localization in the presence of WNT3a , B and C) and nuclear b-catenin (Fig.5, Band E) expression relative to VC cells in the absence of WNT3a, as observed in Figure4, B, C, and E. However, examining VC cells in the presence of WNT3a, there was no significant (p > 0.05) change in the co-localization of NDRG1 and b-catenin relative to the VC cells in the absence of WNT3a (Fig.5, B and F).The overexpression of NDRG1 in cells incubated with WNT3a resulted in significantly (p < 0.001) decreased total bcatenin (Fig.5, B and C) and nuclear b-catenin levels (Fig.5, Band E) versus the VC in the presence of WNT3a.

Figures 4- 6 Figure 5 .Figure 6 .
Figure 5. NDRG1 co-localizes with b-catenin upon NDRG1 overexpression and decreases total b-catenin levels and its nuclear localization in the presence or absence of WNT3a.A-F, NDRG1 overexpression in PANC-1 cells significantly increases the co-localization between b-catenin and NDRG1 in the presence of WNT3a, but particularly its absence.A and B, VC and NDRG1 overexpressing PANC-1 cells were incubated for 24 h/37 C in the presence and absence of WNT3a (100 ng/ml).The cells were then examined for b-catenin (green) and NDRG1 (red) expression and co-localization (yellow) using confocal immunofluorescence microscopy.Cell nuclei were stained by DAPI (blue).Studies were performed using a 60× objective at the same acquisition setting with Olympus Fluoview FV3000 software.Images are representative of three experiments and the quantitative analysis of intensities presented for: (C) b-catenin (reuse from Fig. 4C); (D) NDRG1; (E) nuclear b-catenin (reuse from Fig. 4E); and (F) co-localization between b-catenin and NDRG1.ImageJ software was used for the analysis and the Pearson correlation coefficient (r) was calculated using the Image J plugin, JACoP.Quantitative analyses are presented as the mean ± SD (n = 3).Analysis of pixel intensity and co-localization were performed using 24 cells.The scale bar is 15 mm except for the inset images, where it is 6 mm.Statistical significance is denoted as *p < 0.05, **p < 0.01 and ***p < 0.001 comparing NDRG1 overexpressing PANC-1 cells or VC cells in the presence or absence of WNT3a to VC cells in the absence of WNT3a; ### p < 0.001 comparing NDRG1 overexpressing PANC-1 cells to VC cells in the presence of WNT3; or † † † p < 0.001 comparing NDRG1 overexpressing PANC-1 cells in the absence of WNT3a to NDRG1 overexpressing PANC-1 cells in the presence of WNT3a.

Figure 7 .
Figure 7. Association of PKCa and b-catenin, but also NDRG1 and b-catenin upon NDRG1 overexpression.A-C, NDRG1 and b-catenin and also NDRG1

Figure 8 .
Figure 8. Pharmacological induction of NDRG1 mimics genetic overexpression of this protein leading to the upregulation of PKCa and downregulation of b-catenin expression.PANC-1 PC cells were incubated for 24 h/37 C with control medium, Bp2mT (5 mM), DFO (100 mM) Dp44mT (5 mM), DpC (5 mM), or PPP44mT (5 mM).Total cell protein lysate then underwent SDS-PAGE and was analyzed via western blotting.b-actin was used as a proteinloading control.The blot is representative of three experiments, while the densitometry is mean ± SD (n = 3).Statistical significance is denoted as *p < 0.05, **p < 0.01 and ***p < 0.001 relative to the Control.

Figure 9 .
Figure 9. Schematic demonstrating that overexpression of the metastasis suppressor, NDRG1, inhibits WNT/b-catenin signaling by multi-modal mechanisms involving metabolon formation in PC cells.As an important part of this mechanism, the formation of a metabolon occurs with an association occurring between NDRG1, PKCa, and b-catenin.Expression of NDRG1 and its association with PKCa leads to the stabilization of PKCa that promotes the interaction of PKCa with b-catenin.Due to the latter association, PKCa increases the phosphorylation of b-catenin at Ser33, Ser37, and Thr41, which leads to bcatenin destabilization and degradation.NDRG1 overexpression also decreases GSK-3b protein levels and results in an increase in the inhibitory phosphorylation of GSK-3b (Ser9) and a decrease in the activation phosphorylation of GSK-3b at Tyr216.Levels of the nucleo-cytoplasmic shuttling protein, PAK4, are also decreased by NDRG1 expression, which prevents b-catenin transport to the nucleus.NDRG1 overexpression increases PKCa expression that then reduces AKT activation by decreasing its phosphorylation at Ser473.This latter effect results in the observed decrease in the AKT-mediated phosphorylation of b-catenin at Ser552 that decreasing its transcriptional activity.Together, these multi-modal effector mechanisms of NDRG1 inhibit WNT signaling in PC cells and the expression of the key downstream effector, cyclin D1.
appreciate the award of a Tour de Cure Postgraduate Research Student PhD Scholarship.T. R. and M. G. A. acknowledge the award of Griffith University Postgraduate Research Scholarships (GUPRS).M. D. and D. R. R. (mentor) appreciate support from the Thrasher Research Fund USA Early Career Award.M. D. appreciates a Griffith University Postdoctoral Fellowship from Griffith University and an Elaine Henry Postdoctoral Fellowship from the NBCF.B. K. kindly acknowledges support from The Scientific and Technological Research Council of Turkey (TUBITAK) for a 2219-International Postdoctoral Research Fellowship (app.no.1059B192000031) and Griffith University Postdoctoral Fellowship.